GB2402940A - Biomarkers of inflammation and/or macrophage activation - Google Patents

Abstract

An EMR3 or KMO polypeptide as target in activated-macrophage-related disorders such as inflammatory disorders, and compounds acting as EMR3 or KMO modulators, methods of screening therefore and methods of using them in prevention and treatment of activated-macrophage-related disorders. Also provided are, methods of determining whether macrophages are activated in a sample as well as methods and kits of diagnosis, prognosis, monitoring and assessment of the efficacy of a treatment for activated-macrophage-related disorders using quantification of EMR3 or KMO expression and/or activity levels. The invention relates also to novel disease association of EDG6 or PTGER4 polypeptides and polynucleotides, and to methods of screening for therapeutic agents for the treatment of inflammatory diseases. Antibodies which specifically bind to polypeptides of the invention may be used for the diagnosis of disorders characterized by the over-expression of polypeptide.

Description

À 1 2402940 Genes as a biomarker for macrophaze activation and/or as a

target in inflammation.

Field of the invention:

The present invention is in the field of molecular biology and diagnosis. In particular, the invention relates to an EMR3 or KMO polypeptide as target in activated- macrophage- related disorders such as inflammatory disorders. Said polypeptide may be useful in the prevention and treatment of such disorders. In addition, this polypeptide is a biomarker lo newly found to be associated with macrophage activation, especially with recruited macrophage activation. Such a biomarker may be used in the diagnosis, prognosis, and monitoring of activated-macrophage-related disorders. Such a biomarker may also be useful to assess the efficacy of a treatment for activated-macrophage- related disorders as well as in screening assay to assess the efficiency of an agent to modulate macrophage activation, especially in recruited macrophages.

The present invention relates also to novel disease association of EDG6 or PTGER4 polypeptides and polynucleotides. The invention also relates to novel methods of screening for therapeutic agents for the treatment of inflammatory diseases in mammals.

Antibodies which specifically bind to polypeptides of the invention may be used for the diagnosis of disorders characterized by the over- expression of one polypeptide of the invention.

Background of the invention:

Macrophages can be segregated into two broad groups: resident tissue macrophages and inflammatory recruited macrophages. Tissue macrophages are heterogeneous, and those j; isolated from different anatomical sites, such as liver, kidney or lung, differ in function predi:ably.tbeciuse of adaptive responses to the local micro-environment. Inflammatory recruited macrophages -are derived largely from circulating monocytes, which infiltrate :. ti, damagedti'ssue, but some arise by local cell division. There is now increasing evidence for the heterogeneity of macrophages that have infiltrated inflamed or otherwise damaged 2l tissue, depending on the type and severity of injury, the stage of its evolution and the localization of the macrophages within the tissue.

Macrophages are known to play a central role in the immune response and in inflammation. Macrophages and their released products are indeed involved in inflammation induction and resorption as well as in tissue destruction and subsequent repair. They also display tumoricidal and bactericidal properties. To be able to display those different properties, macrophages need to be activated.

Macrophages are also involved in the development of diseases such as autoimmune disorders and inflammatory diseases. Many clinically significant disorders are indeed accompanied by inflammation, e.g. arthritis, bacterial infections, hypersensitivity, wound, cancer, etc. In active inflammatory bowel disease, there is an increase in the number of mucosal macrophages derived from circulating monocytes. These recruited macrophages are phenotypically different from the resident population of cells and play a major role in mediating the chronic mucosal inflammation seen in patients with ulcerative colitis and Crohn's disease (See Mahida, (2000) Inflamm. Bowel Dis., 6(1):21-33).

Whitney et al. (2000) (Dig. Dis. Sci.; 45(7):1337-42.) report an increased number of gastric mucosal macrophages observed in biopsies of H. pylori-infected versus uninfected children and that this increase is correlated with gastritis severity.

Macrophages are also known to play an important role in rheumatoid arthritis. The abundance and activation of macrophages in the inflamed synovial membrane/pannus significantly correlates with the severity of rheumatoid arthritis (Kinne et al, (2000) Arthritis Res.; 2(3):189-202).

In Experimental autoimmune neuritis, an animal model of the human autoimmune inflammatory disease named Guillain-Barre syndrome, CD4+ T cells mediate demyelination in the peripheral nervous system. Infiltrating macrophages and T cells as well as secretion of cytokines like gamma-interferon are intimately involved in causing pathogenic effects (see Zhu et al., (2001) Exp. Neurol; 169(2):472-8 and Hartung et al, (1990) Ann. Neurol.;27 Suppl:S57-63).

In Lai Invest. (1994);71(4):456-64, Van Goor et al. provide seminal experimental t.

evidences for the crucial contribution of macrophages in the progression of glomerular and interstitial fibrosis.

In J. Neuropathol. Exp. Neurol. !'998);57(2):168-78, Gveric et al. suggest that the activation of the inducible NF-kB pool in macrophages could amplify the inflammatory i reaction seen in multiple sclerosis lesions through up regulation of NF-kB-controlled adhesion molecules and cytokines.

In Pathol. Int. (2000);50(6):441-57, Shiozawa discloses results suggesting that persistent accumulation of macrophages in mesangium induce glomerular sclerosis through expression and activation of matrix metalloproteinases.

Smits et al. (Eur J Clin Invest. (2000);30(6):469-70) describe the clinical and pathological features of both Alzheimer's disease and HIV-1associated dementia and tries to interpret the role of the macrophage and astrocytes therein. Although the neuropathology of Alzheimer's disease and HIV-1-associated dementia differs, Alzheimer's disease being a cortical dementia and HIV-1-associated dementia a subcortical dementia, the process of macrophage activation and the resulting pathways leading to neurotoxicity seem very similar. In both Alzheimer's disease and HIV-1associated dementia, interaction of macrophages and astrocytes appear to play an important role.

Sakai et al., Curr. Opin. Lipidol. (2000);11(5):503-9, suggest that the capacity of oxidized LDL to induce macrophage proliferation in vitro may be involved in the enhanced progression of atherosclerosis in vivo.

Liu et al. (AIDS Res. Hum. Retroviruses (2001);17(15): 1423-33) suggest that cyclooxygenase-2-activated and HIV-1-infected monocyte-macrophages and T cells play a crucial role in the progression of HIV-1 myocarditis to HIV-1 cardiomyopathy.

In Am. J. Respir. Crit. Care Med. (1997);155(3):858-63, Viksman et al. provide evidence for activation of alveolar macrophages, but not peripheral blood monocytes, in subjects with allergic rhinitis and asthma.

In Arch. Pharm. Res. (1999);22(5):437-47, Yoon et al. suggest that macrophages, CD4+ T cells and CD8+ T cells act synergistically to kill the beta cells of Langerhans islets in conjunction with beta cell auto antigens and MHC class I and class 11 antigens, resulting in the onset of autoimmune type I diabetes.

In Am. Rev. Respir. Dis. (1993);147(6 Pt 1):1585-9, Tran Van Nhieu et al. demonstrate the major role of alveolar macrophages in the intra-alveolar production of TNF-alpha, and I they point out the necessity in adult respiratory distress syndrome for a specific intra alveolar therapy.

Chronic obstructive pulmonary disease (COPD) is a chronic, slowly progressive disorder characterized by airflow obstruction (associated with airways inflammation). COPD include emphysema, chronic bronchitis, chronic airflow limitation, chronic airways! obstruction, non-reversible obstructive airways disease, chronic obstructive airways

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disease and chronic obstructive lung disease. The clinical presentation of COPD can vary in severity from simple chronic bronchitis without disability to a severely disabled state with chronic respiratory failure. The diagnosis of COPD is usually suggested by symptoms but can only be established by quantitative measurements, preferably using spirometry (see Thorax, 1997; 52 suppl 5: S1-S32), hereby incorporated by reference in its entirety.

The Global Initiative for Chronic Obstructive Lung Disease recently issued guidelines (Pauwels et al. (2001) Resp. Care 46(8):798-825), hereby incorporated by reference in its entirety that proposed a four-stage classification of COPD severity, namely: Stage 0: At Risk-Characterized by chronic cough and sputum production. Lung function, as measured by spirometry, is still normal.

Stage I: Mild COPD-Characterized by mild airflow limitation (FEV1/FVC < 70% but FEV1 > 80% predicted) and usually, but not always, by chronic cough and sputum production.

Stage II-Moderate COPD: Characterized by worsening airflow limitation (30% < FEV1 < 80% predicted) and usually the progression of symptoms with shortness of breath typically developing on exertion. The division into stages IIA (50% < FEV1 < 80% predicted) and IIB (30% < FEV1 < 50% predicted) is based on the fact that exacerbations are especially seen in patients with a FEV1 below 50% predicted.

Stage III-Severe COPD: Characterized by severe airflow limitation with either; FEV1 < 30% predicted or FEV1 < 50% and the presence of respiratory failure or clinical signs of right heart failure.

FVC is the forced vital capacity and FEV1 the forced expiratory volume in one second as measured by spirometry.

Pulmonary emphysema is a major component of the morbidity and mortality of COPD, a condition that amicts more than 14 million persons in the United States and has become the fourth leading cause of death. In healthy nonsmoker, macrophages comprise the major host cell defense cell in the lower airspace. Cigarette smoking is associated with a more than fivefold increase in total cells recovered by bronchoalveolar ravage (BAL) , with macrophages comprising 95-98%. Moreover, macrophages are prominent in the respiratory bronchioles of cigarette smokers, where emphysematous changes are first manifest. (see Grashoff et al., Am. J. Pathol. (1997); 151(6):1785-90 and Shapiro (1999) Am. J. Respir. Crit. Care Med;160(5 Pt 2):S29-32). ! In Eur. Respir. J., (1999); 14: 245-248, Haslam et al. suggest that the investigatory technique of bronchoalveolar ravage (BAL) has become one of the most valuable research tools for studying inflammatory mechanism in a wide range of diseases that affect the lung and airways in humans and that cytological and microbiological testing of BAL samples are of established value for assisting in clinical diagnosis and management of many lung diseases. These procedure are routinely available in most modern specialist respiratory centers.

Inflammatory and autoimmune diseases are often severely incapacitating, cause the patient great inconvenience and make the patient susceptible to other complications.

Diagnostic and treatment of some auto-immune and inflammatory disorders may be difficult to perform just according to disorder-related symptoms. Providing tools and methods allowing establishing a more precise diagnostic are then very useful.

There is then a real need to find biomarkers of macrophage activation, especially of recruited macrophage activation that can be used for the different purposes including diagnostic and treatment of auto-immune and inflammatory diseases and particularly of lung and airway inflammatory diseases. The results obtained using these biomarkers may be used for diagnostic purposes, alone or in combination with results obtained using other diagnostic methods in order to get a better diagnostic. Such biomarkers may also be useful for prognosis, monitoring, assessing the efficacy of treatment of auto-immune and inflammatory disorders as well as for preventing and treating such diseases.

I) EMR3: EGF-like module containing mucin-like receptor Accession number in the herebelow quoted databases are as follows: Refseq: NM_032571, NM_152939,Unigene: Hs.326777.

In a preferred embodiment of the invention, the cDNA sequence encoding EMR3 is the sequence of SED ID NO: 1, the EMR3 polypeptide sequence is the sequence of SED ID NO: 2.

Epidermal growth factor (EGF)-7-transmembrane (TM7) proteins are Gprotein-coupled receptors (GPCRs) found on monocytes and neutrophils. They include mouse F4/80, mouse and human CD97, and EMR1, the likely human homolog of F4/80. EGF-TM7 glycoproteins have variable numbers of N-terminal EGF-like repeats coupled to a family- B GPCR-related moiety via a mucin-like stalk. By searching DNA databases, using 5 prime and 3-prime RACE, and screening a spleen cDNA library, Stacey et al. (2001) identified cDNAs encoding EMR2, EMR3, and splice variants of each. The deduced 652amino acid EMR3 protein contains an N-terminal signal peptide, 2 EGF-like domains, the first of which does not contain a calcium-binding sequence, a mucin-like spacer region with several N- and O-linked glycosylation sites, a putative GPCR proteolytic site, a TM7 region, and a 51-residue cytoplasmic tail. A truncated EMR3 splice variant containing only the 2 EGF-like domains and no transmembrane domain was predicted to be a soluble protein. Northern blot analysis of tissues and RT-PCR analysis of cell lines revealed that expression of 2.4- and 4.0-kb EMR3 transcripts was restricted to leukocytes. Flow cytometric screening demonstrated that EMR3, but not EMR2, interacts with monocyte derived macrophages and neutrophils activated with HELP but not with CD55, the CD97 ligand. Stacey et al. (2001) noted that EMR3 and EMR2 share a high degree of sequence identity in the TM domains but not in the extracellular domains, whereas EMR2 and CD97 are highly homologous in the EGF-like domains but not in the transmembrane domains, suggesting different ligand-binding and intracellular signaling activities.

II) KMO: kynurenine 3-monooxygenase (kynurenine 3-hydroxYlase) Accession number in the herebelow quoted databases are as follows: RefSeq: NM_003679, SwissProt: Q8CH15.

In a preferred embodiment of the invention, the cDNA sequence encoding KMO is the sequence of SED ID NO: 3, the KMO polypeptide sequence is the sequence of SED ID NO: 4.

Kynurenine 3-monooxygenase (KMO) is an NADPH-dependent flavin monooxygenase that catalyzes the hydroxylation of the L-tryptophan metabolite L-kynurenine to form L 3-hydroxykynurenine.

By screening a human liver cDNA library with a partial Drosophila KMO cDNA, Alberati-Giani et al. (1997) isolated cDNAs encoding human KMO. The predicted 486 amino acid human protein shares 47% sequence identity with Drosophila KMO. When expressed in mammalian cells, recombinant human KMO exhibited kinetic properties similar to those of the native human protein. Northern blot analysis revealed that human KMO is expressed as an approximately 2-kb mRNA in liver and placenta, and at lower levels in kidney. 7/

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By comparing genomic and cDNA sequences, Halford et al. (2001) determined that the KMO gene contains at least 15 exons spanning approximately 68 kb.

III1 EDG6 endothelial differentiation. G-protein coupled receptor 6.

sphingosine 1-phosphate receptor 4 (SIP4). ENDOTHELIAL DlE PERENTIATION GENE 6 Accession number in the herebelow quoted databases are as follows: Refseq: NM_003775, Unigene: Hs.159543.

In a preferred embodiment of the invention, the cDNA sequence encoding EDG6 is the sequence of SED ID NO: 5, the EDG6 polypeptide sequence is the sequence of SED ID NO: 6.

By PCR with degenerate oligonucleotides derived from regions conserved among GPCRs, Grater et al. (1998) cloned differentiated dendritic cell cDNAs encoding EDG6.

The predicted 384-amino acid EDG6 protein has 7 transmembrane domains. The EDG6, protein shares 82% sequence identity with mouse Edg6, 46% identity with human EDG3 44% identity with human EDG1, 39% identity with human EDG4, and 37% identity with human EDG2. Northern blot analysis indicated that EDG6 is expressed as a 1.7-kb transcript in fetal and adult Iymphoid and hematopoietic tissues, lung, and Burkitt Iymphoma cell lines.

IV1 Prostaglandin E2 receptor EP4 subtYpe, Prostanoid EP4 receptor PTGER4.

PROSTAGLANDIN E RECEPTOR 4, EP4 SUBTYPE; PTGER4 Accession number in the herebelow quoted databases are as follows: Refseq: NM_000958, SwissProt: P35408, Unigene: Hs.199248.

In a preferred embodiment of the invention, the cDNA sequence encoding PTGER4 is the sequence of SED ID NO: 7, the PTGER4 polypeptide sequence is the sequence of SED ID NO: 8.

Prostaglandins, thromboxanes, and leukotrienes are oxygenated metabolites of arachidonic acid that are thought to act through 7-transmembrane domain-type receptors, reviewed by Coleman et al. (1994). Prostaglandin E2 activates 4 receptors, designated prostaglandin E receptor subtypes EP1 through EP4. Several cDNAs, originally described as corresponding to an EP2 receptor, were reported by An et al. (1993) and Bastien et al. (1994). The pharmacologically correct EP2 was subsequently isolated by Regan et al. (1994) and the older EP2 sequences were renamed EP4 (Coleman et al., 1994) . See Foord et al. (1996) for a more complete discussion of nomenclature issues.

The EP4 receptor is expressed in a variety of tissues including lung, peripheral blood lymphocytes, and vasculature. Foord et al. (1996) isolated the EP4 receptor gene and showed that it consists of 3 exons and spans about 22 kb of genomic DNA. The first exon is noncoding. Exons 2 and 3 encode a predicted 499-amino acid protein. The gene structure resembles that of the thromboxane receptor, PGI receptor, and PGD receptor.

The promoter region contains motifs found in other cytokine-activated genes. Foord et al. (1996) also observed 2 apparent pseudogenes of EP4R.

Mori et al. (1996) examined the expression of the human EP4 receptor in cells of the immune system. They found that the level of EP4 receptor in T-cell lines was downregulated by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Segi et al. (1998) generated mice deficient in the prostaglandin receptor EP4 by targeted disruption. Loss of the EP4 receptor was not lethal in utero, but most EP4 -/- neonates became pale and lethargic approximately 24 hours after birth and died within 72 hours.

Less than 5% of the EP4 -/- mice survived and grew normally for more than 1 year.

Histologic exam revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization studies showed that EP4 mRNA was strongly expressed in the ductus in wildtype mice. Segi et al. (1998) concluded that neonatal death was at least partly due to patent ductus arteriosus, and that the EP4 receptor plays a role in regulation of the patency of this vessel. Segi et al. (1998) suggested that normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.

Prostaglandin E2 exerts its effects through interaction with specific cell surface receptors.

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin E2 promotes both bone resorption and bone formation. Using homologous recombination, Yoshida et al. (2002) generated mice deficient in each of the 4 EP subtypes to investigate which subtype mediates the bone-forming activity of PGE2 and how activation of this receptor induces bone formation, and identified EP4 as the receptor that mediates bone formation in 9l - \ response to this agent. Consistently, bone formation was induced in wildtype mice by infusion of an EP4-selective agonist and not by agonists specific for other EP subtypes. In cultures of bone marrow cells from wildtype mice, PGE2 induced expression of core- binding factor alpha-1 and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggested that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

Kabashima et al. (2003) demonstrated that although Langerhans cells express all 4 PGE receptor subtypes, their migration to regional lymph nodes was decreased only in EP4- deficient (Ptger4 -/-) mice and in wildtype mice treated with an EP4 antagonist. An EP4 agonist promoted the migration of Langerhans cells, increased their expression of costimulatory molecules, and enhanced their ability to stimulate T cells in the mixed lymphocyte reaction in vitro. Contact hypersensitivity to antigen was impaired in Ptger4 /- mice and in wildtype mice treated with the EP4 antagonist during sensitization.

Kabashima et al. (2003) concluded that PGE2-EP4 signaling thus facilitates initiation of skin immune responses by promoting the migration and maturation of Langerhans cells.

Summary of the invention:

The present invention relates to the use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for inflammatory diseases.

The present invention covers also the use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for macrophage activation.

The present invention encompasses the use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for recruited macrophage activation.

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The present invention recites the use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for an activated-macrophagerelated disorder.

In a prefered embodiment, the present invention relates to the here above quoted use, wherein said activated-macrophage-related disorder is an inflammatory disorder.

In a more prefered embodiment, the present invention encompasses the hereabove quoted use, wherein said inflammatory disorder is a respiratory inflammatory disorder.

In a most prefered embodiment, the present invention covers the use according to the hereabove quoted use, wherein said respiratory inflammatory disorder is chronic obstructive pulmonary disease.

Another embodiment of the present invention is a method of determining whether an individual suffers from CORD, wherein said method comprises the steps of: a) quantifying the macrophagic level of EMR3 or KMO expression and/or activity in a biological sample recovered from said individual; b) determining whether said macrophagic level of EMR3 or KMO expression and/or activity is elevated in said individual compared to the macrophagic level of EMR3 or KMO expression and/or activity in a control sample; and wherein said elevated level of macrophagic EMR3 or KMO expression and/or activity is taken as a sign of the presence of COPD.

A further embodiment of the present invention is a method of screening for therapeutic agents useful in the treatment of inflammatory diseases comprising the steps of: i) contacting a test compound with a polypeptide selected from the group consisting of EDG6, or PTGER4 polypeptides, ii) detect binding of said test compound to said polypeptide.

An additional embodiment of the present invention is a method of screening for therapeutic agents useful in the treatment of inflammation. diseases comprising the steps of: i) determining the activity of a polypeptide selected from the group consisting of EDG6 or PTGER4 polypeptides at a certain concentration of a test compound or in the absence of said test compound, ii) determining the activity of said polypeptide at a different concentration of said test compound.

An additional embodiment of the present invention is a method of diagnosing inflammatory diseases comprising the steps of: i) determining the amount of a polynucleotide selected from the group consisting of EDG6 or PTGER4 polypeptides in a sample taken from said mammal, ii) determining the amount of said polynucleotide in healthy and/or diseased mammals.

Antibodies which specifically bind to polypeptides of the invention are also encompassed and may be used for the diagnosis of disorders characterized by the over-expression of one polypeptide of the invention.

Detailled description of the invention:

I] Definition of terms As used herein, the term << macrophage >> encompasses any type of macrophages including recruited macrophages, or tissue resident macrophages, or a mixture of recruited macrophages and tissue resident macrophages, irrespective of their tissular origin. Preferably, this term refers to recruited macrophages. More preferably, this term refers to recruited macrophages from bronchio-pulmonary origin such as BAL, lung tissue or sputum.

The term "macrophage activation", as used herein, relates to the process by which one or several functions are modulated, enhanced or inhibited in a macrophage or by a macrophage, or in circulating monocytes during their recruitment and differentiation into macrophages, in response to one or several stimuli.

Said functions which are modulated, enhanced or inhibited in a macrophage or in circulating monocytes during their recruitment and differentiation into macrophages are selected from phagocytosis, synthesis and secretion of cytokines, production of proteases, production of angiogenic factors, antigen processing and presentation, and any inflammatory functions.

Said functions which are modulated, enhanced or inhibited by a macrophage are selected from lymphocyte activation, T-cell activation, TH1 or TH2 response, inflammation, fever, anti-infectious activity, anti-tumoral activity, tissue injury, tissue repair, and immune cells attraction.

Said stimuli include any stimulus modulating, enhancing or inhibiting any of the above functions. Illustrating but not limiting examples of such stimuli are aggregation of Fog receptors, lipopolysaccharide (LPS), gammainterferon (g-INF), Migration inhibitory factor (MIF), interleukine 1 (IL1), interleukine 4 (IL-4), interleukine 6 (IL-6), interleukine 10 (IL-10) , Transforming growth factor-beta (TGFb), glucocorticoids and tumor necrosis factor alpha (TNFa).

Preferably, the term << macrophage activation >> relates to the process by which inflammatory functions are modulated, preferably induced or increased, in a macrophage in response to one or several stimuli.

Preferably, said stimuli include any stimulus modulating, preferably inducing or increasing, inflammatory functions in a macrophage. Illustrating but not limiting examples of such stimuli are aggregation of Fog receptors, lipopolysaccharide (LPS), gamma interferon- (g-INF), and Migration inhibitory factor (MIF).

Said inflammatory functions include any function modulated, preferably induced or increased by said stimuli which results in modulating, preferably inducing or increasing an inflammatory reaction. Illustrating but not limiting examples of such inflammatory functions are secretion of inflammatory cytokines, such as interleukines (e.g. interleukine 1 (IL-l), interleukine 6 (I-L6), interleukine 8 (IL-8), interleukine 12 (IL-12), Tumor Necrosis Factor a (TNFa)), leukocyte chemoattractant mediator (LTB4), chemokines (e.g. Monocyte Chemotactic Proteins (MCP-1), macrophage inflammatory protein 1 alpha (MIPla), macrophage inflammatory protein 1 beta (MIPlb), monokine induced by gamma interferon (MIG)), nitric oxide and superoxide production, increase of FcgRl expression and protease liberation.

The term "activated-macrophage-related disorder", as used herein, encompasses all disorders in which the activation of macrophages or activated macrophages play a role such as auto-immune disorders and inflammatory disorders irrespective of the cause of such disorders (e.g. auto-antigen, microorganism infection, hypersensibility, wound, and the like). Illustrating but not limiting examples of such activatedmacrophage-related disorders are inflammatory diseases of the gastrointestinal tract such as Crohn's disease, inflammatory bowel disease, gastritis, colitis, ulcerative colitis, colon irritable, gastric ulcer and duodenal ulcer, inflammatory diseases of the skin such as psoriasis, inflammatory diseases of the respiratory system such as asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD),inflammatory diseases of the musculoskeletal system such as rheumatoid arthritis, or neuritis, renal inflammation, multiple sclerosis, Alzheimer's disease, atherosclerosis and HlV-1-associated dementia, myocarditis, autoimmune diabete, sepsis, septic shock, endotoxic shock, adult respiratory distress syndrome and graft versus host reaction.

Preferably, the term activated-macrophage-related disorder refers to inflammatory disorders including but not limited to inflammatory diseases of the gastrointestinal tract such as Crohn's disease, inflammatory bowel disease, gastritis, colitis, ulcerative colitis, colon irritable gastric ulcer and duodenal ulcer, inflammatory diseases of the skin such as psoriasis, inflammatory diseases of the respiratory system such as asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD), inflammatory diseases of the musculoskeletal system such as rheumatoid arthritis, or neuritis, renal inflammation and myocarditis.

More preferably, the term activated-macrophage-related disorder refers to respiratory inflammatory disorders including but not limited to asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD).

More preferably, the term activated-macrophage-related disorder refers to chronic obstructive pulmonary disease (COPD).

The term "biomarker", as used herein refers to a biological macromolecule which expression and/or activity level is correlated with a biological phenomenon. The term "biomarker for macrophage activation", as used herein, refers to a biological macromolecule that presents expression andlor activity level changes upon macrophage activation. In the context of the invention, this term refers more particularly to a gene which expression and/or activity level increases upon macrophage activation.

Preferably, this term usually refers to a gene which expression and/or activity level increases by at least approximately 1.7 fold to 1000-fold, preferably l0-fold to 50-fold, more preferably x-fold to 50-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20- fold, 30-fold, 50- fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more in activated macrophages compared to non activated macrophages.

This term may also refers to a biological molecule which expression and/or activity level changes upon macrophage activation and preferably also upon monocyte differentiation but not, or to a lesser extent, or not in the same direction during monocyte activation. In particular, this term refers more particularly to a gene which expression and/or activity level increases upon macrophage activation and preferably also upon monocyte differentiationbut not, or to a lesser extent, or decreases in monocyte activation.

The term << EMR3 >>, as used herein, refers to EMR3 expression products, i.e. EMR3 polynucleotides and polypeptides, and fragments thereof.

The term << KMO >>, as used herein, refers to KMO expression products, i. e. KMO polynucleotides and polypeptides, and fragments thereof.

The term << EDG6 >>, as used herein, refers to EDG6 expression products, i.e. EDG6 polynucleotides and polypeptides, and fragments thereof.

The term << PTGER4 >>, as used herein, refers to PIGER4 expression products, i.e. PTGER4 polynucleotides and polypeptides, and fragments thereof.

II] EMR3 or KMO as a biomarker for macrophage activation and a target in inflammation a) Uses of biomarkers The invention relates to the use of EMR3 or KMO as a biomarker for macrophage activation. Particularly, the invention relates to the use of EMR3 or KMO as a biomarker for activatedmacrophage-related disorders, preferably inflammatory disorders, more preferably respiratory inflammatory disorders, even more preferably COPD.

In a first embodiment, it relates to the use of EMR3 or KMO in the in vitro diagnosis, prognosis or monitoring of activated-macrophage-related disorders, preferably inflammatory disorders, more preferably respiratory inflammatory disorders, even more preferably COPD.

In a second embodiment, it relates to the use of EMR3 or KMO in assessing the efficacy of treatment for activated-macrophage-related disorders, preferably inflammatory 15/ disorders, more preferably respiratory inflammatory disorders, even more preferably COPD.

In a third embodiment, it relates to the use of EMR3 or KMO in screening methods and kits.

In a fourth embodiment, said use comprises the use of a EMR3 or KMO polynucleotide, a sequence complementary thereto, or a fragment thereof, in a hybridization or amplification assay and/or the use of compounds able to bind to a EMR3 or KMO polynucleotide, a sequence complementary thereto, or a fragment thereof.

In a fifth embodiment, said use comprises the use of a EMR3 or KMO polypeptide or a fragment thereof, and/or the use of compounds able to bind to a EMR3 or KMO polypeptide or a fragment thereof in protein quantifying assays.

In a sixth embodiment, it relates to the use of EMR3 or KMO in screening assays to determine whether an agent is able to modulate macrophage activation.

b) Method of determining whether macrophages are activated Another object of the invention is a method of determining whether macrophages are activated in a biological sample, said method comprising the steps of: a) quantifying EMR3 or KMO expression and/or activity level in macrophages of said biological sample; and b) determining whether said macrophagic EMR3 or KMO expression and/or activity level in said biological sample is elevated compared to the macrophagic EMR3 or KMO expression and/or activity level in a control sample.

In a first embodiment, said control sample is a sample containing non activated macrophages.

In a second embodiment, said quantifying step comprises quantifying EMR3 or KMO polynucleotides present in macrophages of said biological sample. In a further embodiment, said quantifying step is performed using a hybridization-based assay. In another further embodiment, said quantifying step is performed using an amplification based assay selected from the group consisting of PCR assays, competitive PCR assays, real- time PCR assays, branched DNA-based signal amplification assays, nucleic acid sequence based amplification assays (NASBA), and transcription mediated amplification (TMA). In a preferred embodiment, said amplification is performed using PCR.

In a third embodiment, said quantifying step comprises quantifying EMR3 or KMO polypeptides present in macrophages of said biological sample. In a further embodiment, said quantifying step comprises the use of a compound able to bind to a EMR3 or KMO polypeptide or a fragment thereof. In an even further embodiment, said compound is an antibody or fragment thereof that binds specifically to said EMR3 or KMO polypeptide or fragment thereof.

In a fourth embodiment, said quantifying step comprises quantifying EMR3 or KMO activity in macrophages of said biological sample.

In a fifth embodiment, said biological sample comprises inflammatory, infiltrating or tissue-resident macrophages, preferably inflammatory macrophages from bronchio pulmonary tissues.

Another aspect of the invention is a method of determining whether macrophages are activated in a biological sample, said method comprising the steps of: a) optionally, purifying macrophages from said biological sample; b) extracting nucleic acids from said macrophages; c) contacting said extracted nucleic acids from said macrophages from said biological sample with at least one nucleic acid probe under conditions allowing hybridization to occur between said at least one nucleic acid probe and EMR3 or KMO polynucleotides present in said biological sample in order to form a complex; d) quantifying the amount of hybridized EMR3 or KMO polynucleotides in said biological sample; and e) determining whether said amount of hybridized EMR3 or KMO polynucleotides in said biological sample is elevated compared to the macrophagic EMR3 or KMO expression level in a control sample.

In a first embodiment, said at least one nucleic acid probe is labeled with a detectable molecule. In a second embodiment, said at least one nucleic acid probe has been immobilized on a substrate. In a further embodiment, said at least one nucleic acid probe that has been immobilized on an array. In a third embodiment, said at least one nucleic acid probe has a sequence comprised in a sequence complementary to a EMR3 or KMO polynucleotide.

Still another object of the invention is a method of determining whether macrophages are activated in a biological sample, said method comprising the steps of: a) optionally, purifying macrophages from said biological sample; b) extracting nucleic acids from said macrophages; 17/ : c) contacting said nucleic acids from said macrophages from said biological sample with amplification reaction reagents comprising a pair of amplification primers located on either side of a selected EMR3 or KMO region to be amplified; d) performing an amplification reaction to synthesize EMR3 or KMO amplicons containing said selected EMR3 or KMO region; e) quantifying the amount of said EMR3 or KMO amplicons; and f) determining whether said amount of EMR3 or KMO amplicons in said biological sample is elevated compared to the macrophagic EMR3 OR KMO expression level in a control sample.

Optionally, when the selected EMR3 OR KMO polynucleotide to be amplified is RNA, reverse transcription, and optionally synthesis of a second cDNA strand, are performed prior to said contacting step.

In a first embodiment, said quantifying step comprises hybridization with a labeled probe having a sequence complementary to the EMR3 OR KMO amplicon. In a second embodiment, said quantifying step comprises using incorporated labeled dNTPs, preferably fluorescent dNTPs. In a third embodiment, said quantifying step comprises using a double-stranded DNA specific dye, preferably a fluorescent dye. In a fourth embodiment, said amplification reaction is performed using semi-quantitative PCR or quantitative PCR, preferably real-time PCR.

Still another object of the invention is a method of determining whether macrophages are activated in a biological sample, said method comprising the steps of: a) optionally, purifying macrophages from said biological sample; b) extracting proteins from said macrophages; c) contacting said proteins from said macrophages of said biological sample with a compound able to bind to a EMR3 OR KMO polypeptide or a fragment thereof under conditions allowing said compound to bind to said EMR3 OR KMO polypeptide or said fragment thereof; d) quantifying the amount of said EMR3 OR KMO polypeptides; and e) determining whether said amount of EMR3 OR KMO polypeptides in said biological sample is elevated compared to the macrophagic EMR3 OR KMO expression level in a control sample.

c) Diagnostic, prognosis and monitoring methods for activated-macrophagerelated disorders using quantification of EMR3 or KMO expression and/or activity.

Other objects of the invention are method of diagnosis, prognosis and monitoring of activated-macrophage-related disorder for an individual, wherein said method comprises the step of quantifying the level of macrophagic EMR3 or KMO expression and/or activity levels in at least one biological sample recovered from said individual using any one of the methods described herein.

d) Screening assays for agents modulating macrophage activation Another object of the invention is a method of determining whether an agent interferes with macrophage activation, wherein said method comprises the steps of: a) contacting said agent with macrophages under conditions allowing quantification of EMR3 or KMO expression and/or activity; and b) determining whether EMR3 or KMO expression levels and/or activity are modulated in said macrophages upon macrophage activation using any method described herein.

e) Kits Another aspect of the invention is to provide kits for performing methods according to the invention.

Therefore another object of the invention is a kit, preferably a diagnostic kit for detection 2s of an activated-macrophage-related disorder, wherein said kit comprises reagents to determine the level of EMR3 or KMO expression and/or activity.

In a first embodiment, the invention relates to kits, preferably diagnostic kits for activated-macrophage-related disorders, containing necessary material to quantify the amount of EMR3 or KMO polynucleotides in a biological sample.

fl Methods to find modulators of macrophage activation and uses thereof \ Another object of the invention relates to methods of determining whether a compound is able to modulate the EMR3 or KMO expression and/or activity induced by macrophage activation, said method comprising the steps of: a) contacting said compound with macrophages in a sample under conditions allowing quantification of EMR3 or KMO expression and/or activity; b) activating said macrophages in said sample; and c) determining whether the EMR3 or KMO expression and/or activity level in activated macrophages in said sample changes significantly compared to the macrophagic EMR3 or KMO expression and/or activity level in a control sample containing activated macrophages not contacted with said compound, using any one of the methods disclosed herein.

IIIl Novel disease association of EDG6 or PTGER4 polypeptides and polvaucleotides The EDG6 and PTGER4 polypeptide of the invention are highly expressed in tissues in the states of inflammation. The expression of the EDG6 and PTGER4 polypeptide of the invention in the above mentionned tissues suggests an association between the EDG6 and PTGER4 polypeptide of the invention and inflammatory diseases. The therapeutic regulation of the polypeptides of the invention can be used to treat inflammatory diseases and said EDG6 and PTGER4 polypeptide can be measured to diagnose such diseases.

Of particular importance to the subject invention is the ability to quantitate the level of marker polypeptide as determined by the number of cells associated with a normal or abnormal marker polypeptide level. The number of cells with a particular marker polypeptide phenotype may then be correlated with patient prognosis. In one embodiment of the invention, the marker polypeptide phenotype is determined as a percentage of cells in a biopsy which are found to have abnormally high/low levels of the marker polypeptide. Such expression may be detected by immunohistochemical assays, dot-blot assays, ELISA and the like.

Where tissue samples are employed, immunohistochemical staining may be used to determine the number of cells having the marker polypeptide phenotype. For such A.: staining, a multiblock of tissue is taken from the biopsy or other tissue sample and subjected to proteolytic hydrolysis, employing such agents as protease K or pepsin. In certain embodiments, it may be desirable to isolate a nuclear fraction from the sample cells and detect the level of the marker polypeptide in the nuclear fraction. s

The tissue samples are fixed by treatment with a reagent such as formalin, glutaraldehyde, methanol, or the like. The samples are then incubated with an antibody, preferably a monoclonal antibody, with binding specificity for the marker polypeptides. This antibody may be conjugated to a label for subsequent detection of binding. Samples are incubated for a time sufficient for formation of the immunocomplexes. Binding of the antibody is then detected by virtue of a label conjugated to this antibody. Where the antibody is unlabeled, a second labeled antibody may be employed, e.g., which is specific for the isotype of the anti-marker polypeptide antibody. Examples of labels which may be employed include radionucleotides, fluorescers, chemiluminescers, enzymes and the like.

Examples

Al EMR3 or KMO as a biomarker for macrophage activation and a target in inflammation Example 1: Detection of EMR3 or KMO polynucleotide expression levels in an animal model of acute inflammation LPS-stimulated mice model Male 6-week old C57bl/6 mice are exposed in a plexiglass container to 100 g/ml lipopolysaccharide or LPS (in a 0.9% pyrogen-free physiological sodium chloride solution) aerosol for 1 hour, delivered using the SPAG-2 series 6000 nebuliser system (ICN) with a flow rate of 8 liters per minute at a pressure of 26 + 2 psi. Non-exposed, age-matched littermates are used as controls.

Broncho-alveolar ravages Animals (control and stimulated groups) are then sacrificed 72h after the end of nebulisation to recover cells from broncho-alveolar ravage fluids essentially as follows.

Animals are anesthetized by intra-peritoneal injection of a 0.6% of sodium pentobarbital solution at a dosage of 15 ml/kg. The trachea is cannulated via a tracheostomy. Then, using a syringe, the lungs are rinsed 6 times with 0.3 ml with a sterile PBS solution (PBS without calcium and magnesium), containing 2mM EDTA, warmed at 37 C and the fluid recovered by gentle aspiration. The cell suspension corresponding to 2 mice is collected into conic tubes (Falcon 352096) placed in ice.

Total RNA extraction and cDNA synthesis Collected cells are centrifuged at 4 C. Total RNA is extracted with Trizol (Life Technologies) from 107 cells/prep. Precipitated total RNA (average yield 100,ug) is taken back in 40 pi and analyzed on agarose (1%) gel stained by Ethidium bromide. cDNA synthesis is carried out using Power Script Reverse Transcriptase (Clontech) with 5,ug of total RNA / in 20 Ill preparation mix, corresponding to the suppliers protocol, using random hexamer primers.

Enrichment in macrophages To enrich for the macrophages population, cells recovered from BALs are then passed through a discontinuous Percoll gradient. This discontinuous Percoll gradient is made as follows. A stock solution made with 9 volumes of Percoll (Amersham Pharmacia Biotech APB catalog number 17-0891-01) and 1 volume of 1.5 M NaCI is diluted with 1.5 M NaCI to make two solutions at 1.01 and 1.072 g/ml Percoll using the following formula: Vy = Vj [(p j - p) / (p - py)] Vy = volume of diluting medium in ml.

Vj = volume of stock isotonic Percoll (SIP) in ml.

p j = density of stock isotonic Percoll (SIP) in g/ml = 1.123 g/ml.

py = density of 0.15 M NaCl _ 1.0046 g/ml p = density of diluted solution produced in g/ml To correct for slight variations in volumes and densities of diluting media that affects final density of Percoll solutions, the actual final density of Percoll solutions is measured with a variation of 0,001 g/ml using a densitometer (or a refractometer) or density markers beads (APB - Catalog number: 17-0459-01) as external markers.

The Percoll solutions of 1.01 and 1.072 g/ml respectively are then carefully layered one on the top of the other using, for example, a peristaltic pump set at a low flow rate, in low attachment tubes such as those in Polylabo catalog number 13183.01 starting with the : most dense solution at the bottom of the tube. The preformed gradient is then placed at 4 C.

The cell suspension recovered from BALs is gently layered on the top of the gradient and the cells are separated using a swing bucket head by centrifugation at 300g for 30 minutes at 4 C. Then, the interface is carefully collected with a Pasteur pipette and cells are pelleted by centrifugation (300 g for 30 minutes at 4 C).

Percoll-isolated macrophages harvested from this interface are routinely purified up to 98%, as identified by MGG. Usual yield is between 50 and 70 %.

An optional washing step with saline solution may be performed by centrifugation at 300 g for 10 minutes at 4 C to remove Percoll.

Quantification of EMR3 or KMO expression level EMR3 or KMO expression levels is then quantified using real-time PCR. Real time PCR is performed on a Gene Amp 5700 (Applied Biosystems) using SybrGreen technology (SybrGreen PCR Master Mix from Applied Biosystems, Part Number: 4309155).

Results: Similarly, EMR3 expression was shown to increase in human BAL macrophages (4.5 X average) by COPD patients versus normals.

Similarly, KMO expression was shown to increase in human BAL macrophages (2 X average) by COPD patients versus normals.

B1 Novel disease association of EDG6 or PIGER4 polypeptides and 2s polynucleotides The lung samples are taken by open lung biopsy or by video assisted thoraxoscopy.

Stained samples are obtained. The antibodies used in the study are derived from GPCR peptide sequences.

Tissue sections are deparaffinized, rehydrated and then blocked with 3% H202 in methanol for 30 min followed by antigen retrieval performed with citrate buffer 10 mM pH 6.0 for 5 min in microwave. Tissue sections are then incubated with an antibody diluent with background-reducing components (Dako, Carpinteria, CA) diluted 1/100 in phosphate-buffered saline (PBS) for 45 min. Primary monoclonal anti MMP-8 (20,ug/ml) and MMP9 (5,ug/ml) antibodies (Fuji Chemical Ind., Ltd., Toyama, Japan), and rabbit anti-human myeloperoxidase (Dako, Carpinteria, CA) are used. A secondary biotinylated anti- immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, San Ramon, CA) is used according to the manufacturer's instructions. 3- amino-9-ethyl-carbazole (AEC; BioGenex) in acetate buffer containing 0. 05% H202 is used as substrate. The sections are counterstained with hematoxylin. The primary antibody is replaced by non-immune serum for negative control slides.

Results: 1. EDG6 The staining of bronchial smooth muscle in emphysema patients was strongly positive in contrast to fainter staining in normal tissue.

Respiratory epithelium was strongly positive in allergic polyps of the nasopharynx, as well as in the bronchi of asthma patients; this contrasts with the respiratory epithelium in other diseases as well as normal which were stained more faintly.

2. PTGER4 Respiratory epithelium showed increased staining in bronchitis or emphysema patients samples compared to sample of normal lung.

Samples from patients with emphysema showed increased staining of pneumocytes, alveolar macrophages and seromucous glands. 24/ ,

References: -Stacey, M. et al.,2001, J. Biol. Chem. 276: 18863-18870, -Alberati-Giani, D. et al., 1997, FEBS Lett. 410: 407-412, -Halford, S. et al., 2001, Genomics 72: 203-208, -Graler, M. et al, 1998, Genomics 53: 164-169, -An et al., C1993, Biochem. Biophys. Res. Comm. 197: 263-270, -Bastien et al., 1994,. J. Biol. Chem. 269: 11873-11877, -Coleman et al., Prostaglandins, 1994, 47: 151-168, -Coleman et al., 1994, Pharm. Rev. 46: 205-229, -Foord et al. ,1996, Genomics 35: 182-188, -Kabashima et al., 2003 Nature Med. 9: 744-749, -Mori et al., 1996,. J. Molec. Med. 74: 333-336 -Regan et al., 1994, Molec. Pharm. 46: 213-220, -Segi et al., 1998,Biochem. Biophys. Res. Comm. 246: 7-12, -Yoshida et al., 2002, Proc. Nat. Acad. Sci. 99: 4580-4585.

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Sequence listina: <110> Pfizer limited s <120> Genes as a biomarker for macrophage activation and/or as a target in inflammation <130> 32038 <160> 8 <170> PatentIn version 3.2 <210> 1 <211> 2247 <212> DNA <213> Homo sapiens <400> 1 tUtattgagc taggaaaggt ggttggatta cggcacagta gagagattcc agggctggat 60 ggcgtgggat accagtacca cagaaatgca gggaccattg cttcUtccag gcctctgctt 120 tctgctgagc ctctttggag ctgUgactca gaaaaccaaa acttcctgtg ctaagtgccc 180 cccaaatgct tcctgtgtca ataacactca ctgcacatgc aaccaLggat atacttctgg 240 atctgggcag aaactattca cattcccctt ggagacatgt aacgacatta aLgaatgtac 300 accaccctat agtgtatatt gtggatttaa cgctgUgtgt tacaatgtag aaggaagttt 360 ctactgtcaa tgUgtcccag gatatagact gcattchggg aatgaacaat tcagtaattc 420 caatgagaac acctgtcagg acaccacctc ctcaaagaca accgagggoa ggaaagagct 480 gcaaaagatt gtggacaaat ttgagtcact tctcaccaat cagacUttat ggagaacaga 540 agggagacaa gaaatatcat ccacagctac cactattctc cgggaLgtgg aatcgaaagt 600 tatagaaact gcattgaaag atccagaaca aaaagtcatg aaaatccaaa acgatagtgt 660 agctattgaa actcaagcga ttacagacaa ttgctctgaa gaaagaaaga cattcaactt 720 gaacgEccaa atgaactcaa tggacatccg ttgoagtgac atcatccagg gagacacaca 780 aggtcccagt gccattgcct ttatctcata ttcttctctt ggaaacatca taaatgoaac 840 ttttUttgaa gagaLggata agaaagatca agtgLatctg aactatcagg ttgUgagtgc 900 tgctattgga cccaaaagga acgtgEctat ctccaagtct gtgacgctga chttccagca 960 cgtgaagatg acccccagLa ccaaaaaggt ctEctgtgtc tactggaaga goacagggoa 1020 SS gggoagccag tggtccaggg aLggatgctt cctgatacac gtgaacaaga gtcacaccat 1080 26/ gtgLaattgc agtcacchgt ccagcttcgc tgtcctgatg gccctgacca gccaggagga 1140 ggatcccgtg ctgactgtca tcacctacgt ggggctgagc gtctctctgc tgtgcctcct 1200 cctggcggcc ctcacttttc tcctgtgtaa agccatccag aacaccagoa cctcactgca 1260 tctgcapctc tcgataLgcc tattcctggc ccacctcctc tEcctcgtgg ggattgatag 1320 aactgaaccc aaggtgctgt gctccatcat cgacggtgct ttgcactatc tctacctggc 1380 cgccEtcacc tggatgctgc tggagggtgt gcacctcttc ctcactgoac ggaacctgac 1440 agtggtcaac tactcaagca tcaatagact catgaagtgg atcatgttcc cagtcggcta 1500 IS tggcgUtccc gatgtgactg tggacatttc tgoagactcc tggcctcacc tttatggaac 1560 tgatgatcga tgchggatcc acctggacca gggattcatg tggaghttcc ttggcccagt 1620 ctgtgccatt tCctctgcga atttagtatt gtttatcttg gtchtttgga ttttgaaaag 1680 aaaacUthcc tcactcaata gtgaagtgtc aaccatccag aacacaagga tgchggcEtt 1740 caaagcaaca gctcagctat tcatcatggg ctgoacaLgg tgtctgggct tgctacagOt 1800 gggtccagct gcccaggtca tggcctacct cttcaccatc atcaacagcc tccaaggatt 1860 cttcatcttc ttggtctact gcctcatcag ccagcaggtc cagaaacaat atcaaaagUg 1920 gUttagagag atcgtaaaat caaaatctga gtctgagaca tacacacttt ccagcaagat 1980 gggtcctgac tcaaaaccca gtgaggggga tgtttttcca ggacaagtga agagaaaata 2040 ttaaaactag aatattcaac tccatatgga aaatcatatc catggatctc tttggcatta 2100 tgaagaaLga agctaaggaa aagggaattc attaaacata tcatccttgg agaggaagta 2160 atcaaccUtt acttcccaag ctgEttgttc tccacaatag gatctcaaca aatgtgtggt 2220 aaattgcatt tctcttcaaa aaaaaaa 2247 <210> 2 <211> 652 <212> PRT <213> Homo sapiens <400> 2 Met Gln Gly Pro Leu Leu Leu Pro Gly Leu Cys Phe Leu Leu Ser Leu 1 5 10 15 Phe Gly Ala Val Thr Gln Lys Thr Lys Thr Ser Cys Ala Lys Cys Pro 20 25 30 Pro Asn Ala Ser Cys Val Asn Asn Thr His Cys Thr Cys Asn His Gly 27 ' 40 45 Tyr Thr Ser Gly Ser Gly Gln Lys Leu Phe Thr Phe Pro Leu Glu Thr 50 55 60 Cys Asn Asp.Ile Asn Glu Cys Thr Pro Pro Tyr Ser Val Tyr Cys Gly 70 75 80 Phe Asn Ala Val Cys Tyr Asn Val Glu Gly Ser Phe Tyr Cys Gln Cys 90 95 Val Pro Gly Tyr Arg Leu His Ser Gly Asn Glu Gln Phe Ser Asn Ser 105 110 Asn Glu Asn Thr Cys Gln Asp Thr Thr Ser Ser Lys Thr Thr Glu Gly 120 125 Arg Lys Glu Leu Gln Lys Ile Val Asp Lys Phe Glu Ser Leu Leu Thr 130 135 140 Asn Gln Thr Leu Trp Arg Thr Glu Gly Arg Gln Glu Ile Ser Ser Thr 150 155 160 Ala Thr Thr Ile Leu Arg Asp Val Glu Ser Lys Val Leu Glu Thr Ala 170 175 Leu Lys Asp Pro Glu Gln Lys Val Leu Lys Ile Gln Asn Asp Ser Val 185 190 Ala Ile Glu Thr Gln Ala Ile Thr Asp Asn Cys Ser Glu Glu Arg Lys 200 205 Thr Phe Asn Leu Asn Val Gln Met Asn Ser Met Asp Ile Arg Cys Ser 210 215 220 Asp Ile Ile Gln Gly Asp Thr Gln Gly Pro Ser Ala Ile Ala Phe Ile 225 230 235 240 Ser Tyr Ser Ser Leu Gly Asn Ile Ile Asn Ala Thr Phe Phe Glu Glu 245 250 255 Met Asp Lys Lys Asp Gln Val Tyr Leu Asn Ser Gln Val Val Ser Ala 260 265 270 Ala Ile Gly Pro Lys Arg Asn Val Ser Leu Ser Lys Ser Val Thr Leu 275 280 285 s Thr Phe Gln His Val Lys Met Thr Pro Ser Thr Lys Lys Val Phe Cys 290 295 300 Val Tyr Trp Lys Ser Thr Gly Gln Gly Ser Gln Trp Ser Arg Asp Gly 305 310 315 320 Cys Phe Leu Ile His Val Asn Lys Ser His Thr Met Cys Asn Cys Ser 325 330 335 His Leu Ser Ser Phe Ala Val Leu Met Ala Leu Thr Ser Gln Glu Glu 340 345 350 ou Asp Pro Val Leu Thr Val Ile Thr Tyr Val Gly Leu Ser Val Ser Leu 355 360 365 Leu Cys Leu Leu Leu Ala Ala Leu Thr Phe Leu Leu Cys Lys Ala Ile 370 375 380 Gln Asn Thr Ser Thr Ser Leu His Leu Gln Leu Ser Leu Cys Leu Phe 385 390 395 400 Leu Ala His Leu Leu Phe Leu Val Gly Ile Asp Arg Thr Glu Pro Lys 405 410 415 Val Leu Cys Ser Ile Ile Ala Gly Ala Leu His Tyr Leu Tyr Leu Ala 420 425 430 Ala Phe Thr Trp Met Leu Leu Glu Gly Val His Leu Phe Leu Thr Ala 435 440 445 Arg Asn Leu Thr Val Val Asn Tyr Ser Ser Ile Asn Arg Leu Met Lys 450 455 460 Trp Ile Met Phe Pro Val Gly Tyr Gly Val Pro Ala Val Thr Val Ala 465 470 475 480 Ile Ser Ala Ala Ser Trp Pro His Leu Tyr Gly Thr Ala Asp Arg Cys 485 490 495 Trp Leu His Leu Asp Gln Gly Phe Met Tr Ser Phe Leu Gly Pro Val ) 29i r 500 505 510 Cys Ala Ile PheSer Ala Asn Leu Val Leu Phe Ile Leu Val Phe Trp s 515 520 525 Ile Leu Lys Arg Lys Leu Ser Ser Leu Asn Ser Glu Val Ser Thr Ile 0 530 535 540 Gln Asn Thr Arg Met Leu Ala Phe Lys Ala Thr Ala Gln Leu Phe Ile 545 550 555 560 Leu Gly Cys Thr Trp Cys Leu Gly Leu Leu Gln Val Gly Pro Ala Ala 565 570 575 Gln Val Met Ala Tyr Leu Phe Thr Ile Ile Asn Ser Leu Gln Gly Phe 580 585 590 Phe Ile Phe Leu Val Tyr Cys Leu Leu Ser Gln Gln Val Gln Lys Gln 595 600 605 Tyr Gln Lys Trp Phe Arg Glu Ile Val Lys Ser Lys Ser Glu Ser Glu 610 615 620 Thr Tyr Thr Leu Ser Ser Lys Met Gly Pro Asp Ser Lys Pro Ser Glu 625 630 635 640 Gly Asp Val Phe Pro Gly Gln Val Lys Arg Lys Tyr 645 650 <210> 3 <211> 5000 <212> DNA <213> Homo sapiens <400> 3 ggcacgaUca gaaDcaacaa taattgtgaa aaatacUtca gcagttatgg actcatctgt 60 cattcaaagg aaaaaagLag ctgtcattgg tOgUggattg gttggctcat tacaagcaLg 120 cEttcttgca aagaggaatt tccagattga tOtataLgaa gctagggaag atactcgagt 180 ggctaccUtc acacgtOgaa gaagcattaa cttagccctt tctcatagag gacgacaagc 240 SS cEtgaaaDct gttggcctgg aagatcagat tgtatcccaa ggLattccca tgagagcaag 300 aaLgatccac tatctEtcag gaaaaaagtc tgcaatEccc tatgOgacaa agtctcagta 360 30/ tattctttct gtaagoagag aaaatctaaa caaggatcta ttgactgatg ctgagaaata 420 ccccaatgtg aaaatgcact ttaaccacag gctgttgaaa tgtaatccag aggaaggaat 480 gatcacagtg cttggatatg acaaagttcc caaagatgtc acttgtgacc tcattgLagg 540 atgtgatgga gcctattcaa ctgtcagatc tcacctgaLg aagaaacatc gctttgatta 600 cagtcagcag tacattcctc atgggLacat ggagUtgact attccaccta agaacggaga 660 ttaLgccatg gaacctaatt atatgcatat ttggcctaga aataccttta tgatgattgc 720 acttcctaac atgaacaaat catEcacatg tactttgttc atgcccUttg aagagtttga 780 aaaactEcta accagtaaLg atgUggtaga tttcttccag aaatactttc cggatgacat 840 ccctctaatt ggagagaaac tcctagtgca agatttctCc chghtgcctg cccagcccat 900 gatatctgta aagtgatatt catttcactt taaatctcac tgtgtactgc tgggagatgc 960 agctcatgct atagtgacgt tttttgggca aggaatgaat gcgggcthtg aagactgcht 1020 ggtatttgat gagttaatgg ataaattcag taacpacatt agtttgtgtc ttcctgtgtt 1080 ctcaagattg agaatcccag atgatcacgc gatttcagac ctatccatgt acaattacat 1140 agagatgcga gcacaLgtca actcaagatg gttcatEttt cagaagaaca tggagagatt 1200 tottcaLgcg attatgccat cgaccEttat cactctctat acaatggtca ctUtttccag 1260 aataagatac catgaggaLg tgcagaghtg gcattggcaa aaaaaggtga taaacaaagg 1320 actctttttc ttgggatcac tgatagacat cagoagLacc tacatacUta tacactacat 1380 gtcaccacga tcthtcatct gcttgagaag accatggaac tggatagctc acttccggaa 1440 tacaacatgt ttccacgoaa aggccgtgga ctccctagaa caaatttcca atctcattag 1500 caggtgatag aaaggthttg tggtagcaaa tgcaLgattt ctctgtgacc aaaattaaDc 1560 aLgaaaaaaa tgtttccatt gccatatttg attcactagt ggaagatagt gtCctgctta 1620 taattaaact gaatgtagag tatatctgta tgttaattgc aattactggt tggggggtgc 1680 atUttaaaag atgaaacatg cagattccct acattacaca cactcaggtt gagtcattct 1740 aactataaaa gtgcaatgac taagatcctt cacttatctg aaagtaaggc cctagaLgac 1800 tcagggaaga cagLaatcat gcctUtCcUt taaaagacac aataggactc gcaacagcat 1860 tgactcaaca cataggacta aaaatcacaa cttaactagc aLgttaactg cactttCcat 1920 tacgtgaatg gaacUtacct aaccacaggg ctcagachta ctagataaaa ccagaaaLgg 1980 aaataaggaa ttcaggggag ttccagagac ttacaaaatg aactcattEt attUtcccac 2040 cttcaaatat aagLattatc atctatatgt ttatcgEcta tctatctatc atctatatat 2100 ctatctatca tctatctatc tatctatcta tctatotatc tatctatata tctatctatc 2160 tatatttatt tatgLattta gagatcaggt ctcactctgt tgaccaggct ggagtgcagt 2220 ggtgagatct gggttcactg caacctctgc ctcctgggat caagcaatcc tcccacttca 2280 gcatcccaaa tagctggggc taccatggta tttttcagta gagaccgggt cttgccatgc 2340 tgcccaggcc agtatcaaac tcctggcctc atgtgatctg cccacctcag catcccaaag 2400 tacagggatt agagttgtga gccaccgctg ccagaccaga ghtaccatct aaagataaga 2460 aaaaggctat taatatcata ctaagtgaag gacaggaaag ggttttattc ataaattaaa 2520 tgtctacatg tgccagaatg gaaaggaaac aaggggagac aacttttata gaaatacaaa 2580 gacattactt tattcaattt cagaccatca gaagoaattt actaatttat tcttcgacta 2640 catactgcag cagaaccagc aatacacttg attUttaaaa gcacatttag tgaaatgttt 2700 tcEttggttc atccttchtt aacaggctgc tgagtcactc agaaatcctt caaacatgat 2760 taattatgaa gatgaaacac tagagtcata taagaaataa aaattgggca ataaaataaa 2820 aLgattcagt gtUtctUttc tatattgtca atgaaaacat tgagtCctaa taatccatgt 2880 tcagtttgta gggaaagaaa aaataathtt tccttatacc cactUtaggt tccttggctg 2940 gggcccatat aacaaaagac agattgacaa gagaaaaaca aacataaatt tattagaggg 3000 tatatgtaat atatatgtgg gaaatacagg ggaatgagca aatctcaaag agatggagtc 3060 ttagaactcc ctggattata tagcatcgac aaaqaacagt aaatttttag agaaacaaca 3120 aaacaaagaa aaagagattt gagtctgtag gggcagcaat ttgggggaag caaatatatg 3180 ggagtUtgcc ttgtagattc ctctggtggt ggtctccagg ctgacaagga ttcaaagUtg 3240 tctchgaaac tcctctttgt catactgoac atataaaacg tcttttgtht ccaacaagag 3300 gatttctttt tcattataga attatctcct tgataacUtg atcagatata ggacatgaca 3360 ctgaatagag tccaacagta caaaaaaaat tcagtatgEt ctagctactt cacacaLgtg 3420 tacgagacag ttatttttac agtaaggLat thtcgagaaa aatgcattac gtgtUttgga 3480 _ aaatagagta atttaaaaaa tatatttgea atgaaaatct ccaacacatt agaagatgat 3540 gatghtagat gcccatagtg tgccacaagt ggUtttEtca ttaLgtaaag cacccgttga 3600 50: attaaaagaa tttgtUtUtg ttcaacctct tcctgaggcc caagagcata tgggcaattc 3660 ggattUcctg ctggaccaca aggttctgtt gatattacat agaaacgggt attccagaca 3720 ctCcttatga tgaaagtcca aaagUggcat ccaatttaag gcaccatctt tcgttgccat 3780 tctEcattcc tacaaaggac gaacUtggat tacatcaact ttggacccat tggttttgtc 3840 gctgtcgtca actgacagtg attcaccact ggtgatgata aaaatgatgg aagaagaght 3900 gaaagtcact tUtUtatttg gcctgtcccc atctttctgt gacatcacaa tgggEctgat 3960 ctgcatttca cUtccagctg ctggLaggtc tttagcaggc ctctggoacc tcagcagtcg 4020 gaggcacaga agaLgcaaaa gggatattcg aaactgggoa gagaaaaaat aaagtggaat 4080 attaagtaaa agttgggoac taatctggat taacattcga ggaaatcagt tgagctgatt 4140 taagttghtt tttgtttgtt agcaggtgtg gatgtggggt tatgtggtca tgctcagatc 4200 tacctaaatc acaccagagc tttatgtctt ttathcatEc taaatattat taaccOgaat 4260 IS atgtaggacc atttcaatac cttgtaatcc tccaagcttc aatctgoaca cactttatat 4320 gagggcaggt acaactatta agagatEttg aacattaagt tagtccacaa atattcagtg 4380 ggcatctact aggtgacagc cactgtgcta taattagaga ctttttacta taapcatcaa 4440 aaacagataa ggatcttcct ggcagagEtt acagcatggt gtacttgcta atgtctcttt 4500 aattaggtga agaatttttt tttUctatcg aaattactaa tcagttgggg aaaaaaatac 4560 tatagcagac agoactaaLg tcatcaacaa acattgttct tatccgtgtc ctgggtacaa 4620 catcgaataa tatttcttgg cctccttUcc gcttctcatc tctgctgEtc ctctctacaa 4680 gaacctggga ggccaaegcc taaagatcat aatatcacaa tggaaggaac ctagattcct 4740 aaatgactgc ataggacaga tcccatctcc tccacacaat acattattag achgaactgt 4800 gacctgaaat gagcaataaa ctctgtatta attcactgaa atgttOggOt tgattgttat 4860 agtagtcggt ccatcatgac cagLaaaaca taaatcaaaa gttaatgtaa ttgttatccc 4920 attatttaga gcgaaataaa tgttgaatat atggactttc tcagattagg aaataccaat 4980 taaaaatata ataaatagct 5000 <210> 4 <211> 486 <212> PRT <213> Homo sapiens <400> 4 Met Asp Ser Ser Val Ile Gln Arg Lys Lys Val Ala Val Ile Gly Gly 1 5 10 15 Gly Leu Val Gly Ser Leu Gln Ala Cys Phe Leu Ala Lys Arg Asn Phe 25 30 Gln Ile Asp Val Tyr Glu Ala Arg Glu Asp Thr Arg Val Ala Thr Phe 40 45 Thr Arg Gly Arg Ser Ile Asn Leu Ala Leu Ser His Arg Gly Arg Gln 55 60 s Ala Leu Lys Ala Val Gly Leu Glu Asp Gln Ile Val Ser Gln Gly Ile 70 75 80 Pro Met Arg Ala Arg Met Ile His Ser Leu Ser Gly Lys Lys Ser Ala 90 95 Ile Pro Tyr Gly Thr Lys Ser Gln Tyr Ile Leu Ser Val Ser Arg Glu 105 110 Asn Leu Asn Lys Asp Leu Leu Thr Ala Ala Glu Lys Tyr Pro Asn Val 115 120 125 Lys Met His Phe Asn His Arg Leu Leu Lys Cys Asn Pro Glu Glu Gly 135 140 Met Ile Thr Val Leu Gly Ser Asp Lys Val Pro Lys Asp Val Thr Cys 150 155 160 Asp Leu Ile Val Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ser His 170 175 Leu Met Lys Lys Pro Arg Phe Asp Tyr Ser Gln Gln Tyr Ile Pro His 185 190 Gly Tyr Met Glu Leu Thr Ile Pro Pro Lys Asn Gly Asp Tyr Ala Met 195 200 205 Glu Pro Asn Tyr Leu His Ile Trp Pro Arg Asn Thr Phe Met Met Ile 210 215 220 Ala Leu Pro Asn Met Asn Lys Ser Phe Thr Cys Thr Leu Phe Met Pro 225 230 235 240 Phe Glu Glu Phe Glu Lys Leu Leu Thr Ser Asn Asp Val Val Asp Phe 245 250 255 SS Phe Gln Lys Tyr Phe Pro Asp Ala Ile Pro Leu Ile Gly Glu Lys Leu 260 265 270

-

Leu Val Gln Asp Phe Phe Leu Leu Pro Ala Gln Pro Met Ile Ser Val 275 280 285 Lys Cys Ser Ser Phe His Phe Lys Ser His Cys Val Leu Leu Gly Asp 290 295 300 Ala Ala His Ala Ile Val Pro Phe Phe Gly Gln Gly Met Asn Ala Gly 0 305 310 315 320 Phe Glu Asp Cys Leu Val Phe Asp Glu Leu Met Asp Lys Phe Ser Asn 325 330 335 Asp Leu Ser Leu Cys Leu Pro Val Phe Ser Arg Leu Arg Ile Pro Asp 340 345 350 Asp His Ala Ile Ser Asp Leu Ser Met Tyr Asn Tyr Ile Glu Met Arg 355 360 365 Ala His Val Asn Ser Ser Trp Phe Ile Phe Gln Lys Asn Met Glu Arg 370 375 380 Phe Leu His Ala Ile Met Pro Ser Thr Phe Ile Pro Leu Tyr Thr Met 385 390 395 400 Val Thr Phe Ser Arg Ile Arg Tyr His Glu Ala Val Gln Arg Trp His 405 410 415 Trp Gln Lys Lys Val Ile Asn Lys Gly Leu Phe Phe Leu Gly Ser Leu 420 425 430 Ile Ala Ile Ser Ser Thr Tyr Leu Leu Ile His Tyr Met Ser Pro Arg 435 440 445 Ser Phe Leu Cys Leu Arg Arg Pro Trp Asn Trp Ile Ala His Phe Arg 450 455 460 Asn Thr Thr Cys Phe Pro Ala Lys Ala Val Asp Ser Leu Glu Gln Ile 465 470 475 480 Ser Asn Leu Ile Ser Arg <210> 5 <211> 1566 t: <212> DNA <213> Homo saplens <400> 5 gagtcagccc ccgggggagg ccatgaacgc cacggggacc ccggtggacc cagagtcctg60 ccaacagctg gaggccggcg ggcacagcag gctcattgUt ctgcactaca accactcggg120 ccggctggac gggagagggg ggccggagga tggaggcctg ggggccctgc gggggctgtc180 ggtggacgcc agatgcctgg tggtgctgga gaacttgctg gUgctggcgg ccatcaccag240 ccacatgcgg tcgagacgct gggtctacta ttgactggtg aacatcaegc tgagtgacct300 gctcacgggc gcggcatacc tggccaacgt gctgctgtag ggggcccgca ccttccgtat360 ggcgcccgcc cagtggEtcc tacgggaggg cctgctcEtc accgccctgg ccgactccac420 cttcagcctg ctattcactg caggggagcg ctttgccacc atggtgaggc cggtggacga480 gagcggggcc accaagacca gccgcgtcta cggctCcatc ggcctctgat ggctgctggc540 cgcgctgctg gggatgatgc ctttgatggg ctggaactgc ctgtgcgact ttgaccgctg600 ctccagcctt ctgcccctct actccaagag ctacatcctc ttctgcctgg tgatcttcgc660 cggcgEcctg gccaccatca tgggcctcta tggggccatc tEccgactgg tgcaggacag720 cgggcagaag gcaccacgcc cagcggcacg ccgcaaggcc cgccgcctgc tgaagacggt780 gctgaLgatc ctgctggcat tcctggtgtg ctggggcaca ctctCcgggc tgctgctggc840 cgacgtcttt ggctccaacc tctgggccca ggagtacctg cggggcatgg actggatcct900 ggccctggcc gtcctcaact cggaggtcaa ccacatcatc tactcattcc gcagcaggga960 ggtgtgoaga gacgtgctca gcttcatatg ctgcgggtgt ctccggctgg gcatgcgagg 1020 gcccggggac tgcctggacc gggcagtaga ggctcactcc ggagatCcca ccaccgacag 1080 ctctctgagg ccaagggaca gctttcgcgg ctcccgctcg ctcagctttc ggatgcggga 1140 gcccctgtcc agcatctcca gcgtgcggag catctgaagt tgoagtcttg cgtgtggatg 1200 gtgcagccac cgggtgagtg ccaggcaggc cctcchgggg tacaggaagc tgtgtgcacg 1260 cagcctcgcc tgLaLgggga gcagggaacg ggacaggccc ccaLggtcht cccggtggcc 1320 tctcggggct tatgacgcca aatgggcttc ccaLggtcac catggacaag gaggtaacca 1380 caccacctcc ccgtaggagc agagagcacc ctggtgtggg ggcgagtggt tccccacaac 1440 cccgctUctg tgtgattctg gggaagtccc ggcccctctc tgggcctcag tagggctccc 1500 SS aggctgcaag gggtggactg tgggaLgoat gccctggcaa cattgaagtt cgatcatggt 1560 aaaaaa 1566 <210> 6 <211> 384 <212> PRT <213> Homo sapiens <400> 6 Met Asn Ala Thr Gly Thr Pro Val Ala Pro Glu Ser Cys Gln Gln Leu Ala Ala Gly Gly His Ser Arg Leu Ile Val Leu His Tyr Asn His Ser 25 30 Gly Arg Leu Ala Gly Arg Gly Gly Pro Glu Asp Gly Gly Leu Gly Ala 40 45 Leu Arg Gly Leu Ser Val Ala Ala Ser Cys Leu Val Val Leu Glu Asn 55 60 Leu Leu Val Leu Ala Ala Ile Thr Ser His Met Arg Ser Arg Arg Trp 70 75 80 Val Tyr Tyr Cys Leu Val Asn Ile Thr Leu Ser Asp Leu Leu Thr Gly Ala Ala Tyr Leu Ala Asn Val Leu Leu Ser Gly Ala Arg Thr Phe Arg 105 110 Leu Ala Pro Ala Gln Trp Phe Leu Arg Glu Gly Leu Leu Phe Thr Ala 120 125 Leu Ala Ala Ser Thr Phe Ser Leu Leu Phe Thr Ala Gly Glu Arg Phe 135 140 Ala Thr Met Val Arg Pro Val Ala Glu Ser Gly Ala Thr Lys Thr Ser 150 155 160 Arg Val Tyr Gly Phe Ile Gly Leu Cys Trp Leu Leu Ala Ala Leu Leu 165 170 175 Gly Met Leu Pro Leu Leu Gly Trp Asn Cys Leu Cys Ala Phe Asp Arg 185 190 Cys Ser Ser Leu Leu Pro Leu Tyr Ser Lys Arg Tyr Ile Leu Phe Cys 200 205 Leu Val Ile Phe Ala Gly Val Leu Ala Thr Ile Met Gly Leu Tyr Gly 210 215 220 Ala Ile Phe Arg Leu Val Gln Ala Ser Gly Gln Lys Ala Pro Arg Pro 225 230 235 240 Ala Ala Arg Arg Lys Ala Arg Arg Leu Leu Lys Thr Val Leu Met Ile 245 250 255 Leu Leu Ala Phe Leu Val Cys Trp Gly Pro Leu Phe Gly Leu Leu Leu 260 265 270 Ala Asp Val Phe Gly Ser Asn Leu Trp Ala Gln Glu Tyr Leu Arg Gly 275 280 285 Met Asp Trp Ile Leu Ala Leu Ala Val Leu Asn Ser Ala Val Asn Pro 290 295 300 Ile Ile Tyr Ser Phe Arg Ser Arg Glu Val Cys Arg Ala Val Leu Ser 305 310 315 320 Phe Leu Cys Cys Gly Cys Leu Arg Leu Gly Met Arg Gly Pro Gly Asp 325 330 335 Cys Leu Ala Arg Ala Val Glu Ala His Ser Gly Ala Ser Thr Thr Asp 340 345 350 Ser Ser Leu Arg Pro Arg Asp Ser Phe Arg Gly Ser Arg Ser Leu Ser 355 360 365 Phe Arg Met Arg Glu Pro Leu Ser Ser Ile Ser Ser Val Arg Ser Ile 370 375 380 <210> 7 c211> 2052 <212> DNA SO <213> Homo sapiens <400> 7 cgggagcgga cagLaagccc gOcagacaga gaggaagatg aacaDcccca ggoagagact 60 ctcggagagt ggacaccgag ccgccaccag gtaDccagga gccgcctcag cgOcaUccOc 120 aaactcagta googacagtg ctgcoagtgg ctUgggcgag ggaDcccaga gchgOggacc 130 aaggctccgc cacctgcggg acagcctcac accUaacgct gtcctcccgc agacgagacc 240 ggcgggcact gcaaaDctgg gactcgtctt tgaaggaaaa aaaatagcga gtaagaaatc 300 cagcaccatt cttcactgac ccatcccgct gcacctcttg tEtcccaagt ttttgaaagc 360 tggcaactct gacctcggtg tccaaaaatc gacaUccact gagacaggct ttgagaagcc 420 gaagatttgg cagUttccag actgagcagg aacaagUtga aaDcaggttg gaggcgggtc 480 caggacatct gagggctgag gOctgOgggg tagUgagOct gccaccgctc gUgccgctac 540 agacccaDcc ttgcactcca aggatgcOca ccDccagcca ctatcatgtc cactcccOgg 600 gtcaattcgt ccDcatcctt gapccccOac cOgctgaaca gcccagtgac catcccggcg 660 gtgatgttca tattagggOt ggtgggcaac chggUggcca tagtggUgct gUgoaagtcg 720 cgoaaggaDc agaaggagac gacattctac acDctggtat gtggOctggc tgtcaccgac 780 ctOttOggca ctttgttOgt gaDccaggtg accatcDcca cgtacatgaa gggacaatgg 840 cccgggggcc aDccOctgUg cOagtacaDc accUtcatCc tgctattatt cagcctgtct 900 ggcatcagca tcatctgcDc catgagUgtc gagcOctacc tggccatcaa ccatgcctat 960 ttctacagcc actacgtgga caaDcgattg gagggcctca cgatctttgc agtctatgag 1020 tccaacUtgc tcttttgcgc gctgaccaac atOgOtctcg gtaDctcgcg gctgcagtac 1080 ccagacacct ggtgattcat cgactggacc accaacOtga cggcgcacOc cgactactcc 1140 tacaLgLacg cgggcttcag ctccttcctc athctcDcca ccgtcctctg caacgtgctt 1200 gtgtgcggcg cgctgctcag catgoaccgc cagttcatgc gccDcacctc gctgggcacc 1260 gagcagoacc acOcOgaegc gOccgcctcg ghUgcctccc ggggccaccc cgctgactcc 1320 ccagccttgc cDcDcatcag cUactttcgg cgccDccgga gattccgcog catcgcgggc 1380 gacgagatcc agatggtcat cttactcatt gccacatccc tggUggtgat catctgctcc 1440 atcccgctag tggtgcgagt gtUcgtcaac cagttatatc aDccaagttt ggagcgagaa 1500 gtcagLaaaa atccagattt goaggacatc cgaattgctt ctgtgaaccc catcctagac 1560 ccctggatat atatcctcct gagaaagaca gUgctcagta aagcaataga gaagatcaaa 1620 tgcctcEtct gccOcattgg cgOgtccaDc agOgagcgat ccggacagca ctgctcagac 1680 agtcaaagga catcEtctgc caLgtcaggc cactctcDct catCcatatc ccgggagctg 1740 aaggagatca goagLacatc tcagaccctc ctgccagacc tatcactgcc agacctcagt 1800 gaaaatggcc ttOgaggoag gaatttgctt ccaggUgtgc ctgOcaLggg cctggoccag 1860 gaagacacca catcactgag gactttgcDa atatcagaga cctcagactc ttcacagggt 1920 caggactcag agagtgtctt actggtggat gaggctggtg ggagcggcag ggcttgggcc 1980 tgccctaagg ggagctccct gcaagtcaca tttcccagtg aaacactgaa cEtatcagaa 2040 aaatgtatat aa 2052 <210> 8 0 <211> 488 <212> PRT <213> Homo sapiens <400> 8 Met Ser Thr Pro Gly Val Asn Ser Ser Ala Ser Leu Ser Pro Asp Arg 1 5 10 15 Leu Asn Ser Pro Val Thr Ile Pro Ala Val Met Phe Ile Phe Gly Val 25 30 Val Gly Asn Leu Val Ala Ile Val Val Leu Cys Lys Ser Arg Lys Glu 35 40 45 Gln Lys Glu Thr Thr Phe Tyr Thr Leu Val Cys Gly Leu Ala Val Thr 55 60 Asp Leu Leu Gly Thr Leu Leu Val Ser Pro Val Thr Ile Ala Thr Tyr 70 75 80 Met Lys Gly Gln Trp Pro Gly Gly Gln Pro Leu Cys Glu Tyr Ser Thr 90 95 Phe Ile Leu Leu Phe Phe Ser Leu Ser Gly Leu Ser Ile Ile Cys Ala 105 110 Met Ser Val Glu Arg Tyr Leu Ala Ile Asn His Ala Tyr Phe Tyr Ser 115 120 125 His Tyr Val Asp Lys Arg Leu Ala Gly Leu Thr Leu Phe Ala Val Tyr 135 140 Ala Ser Asn Val Leu Phe Cys Ala Leu Pro Asn Met Gly Leu Gly Ser 150 155 160 Ser Arg Leu Gln Tyr Pro Asp Thr Trp Cys Phe Ile Asp Trp Thr Thr 170 175 Asn Val Thr Ala His Ala Ala Tyr Ser Tyr Met Tyr Ala Gly Phe Ser 185 190 s Ser Phe Leu Ile Leu Ala Thr Val Leu Cys Asn Val Leu Val Cys Gly 200 205 Ala Leu Leu Arg Met His Arg Gln Phe Met Arg Arg Thr Ser Leu Gly 210 215 220 Thr Glu Gln His His Ala Ala Ala Ala Ala Ser Val Ala Ser Arg Gly 225 230 235 240 His Pro Ala Ala Ser Pro Ala Leu Pro Arg Leu Ser Asp Phe Arg Arg 245 250 255 Arg Arg Ser Phe Arg Arg Ile Ala Gly Ala Glu Ile Gln Met Val Ile 260 265 270 Leu Leu Ile Ala Thr Ser Leu Val Val Leu Ile Cys Ser Ile Pro Leu 275 280 285 Val Val Arg Val Phe Val Asn Gln Leu Tyr Gln Pro Ser Leu Glu Arg 290 295 300 Glu Val Ser Lys Asn Pro Asp Leu Gln Ala Ile Arg Ile Ala Ser Val 305 310 315 320 Asn Pro Ile Leu Asp Pro Trp Ile Tyr Ile Leu Leu Arg Lys Thr Val 325 330 335 Leu Ser Lys Ala Ile Glu Lys Ile Lys Cys Leu Phe Cys Arg Ile Gly 340 345 350 Gly Ser Arg Arg Glu Arg Ser Gly Gln His Cys Ser Asp Ser Gln Arg 355 360 365 Thr Ser Ser Ala Met Ser Gly His Ser Arg Ser Phe Ile Ser Arg Glu 370 375 380 Leu Lys Glu Ile Ser Ser Thr Ser Gln Thr Leu Leu Pro Asp Leu Ser 385 390 395 400 Leu Pro Asp Leu Ser Glu Asn Gly Leu Gly Gly Arg Asn Leu Leu Pro ) 405 410 415 Gly Val Pro Gly Met Gly Leu Ala Gln Glu Asp Thr Thr Ser Leu Arg 420 425 430 Thr Leu Arg Ile Ser Glu Thr Ser Asp Ser Ser Gln Gly Gln Asp Ser 435 440 445 Glu Ser Val Leu Leu Val Asp Glu Ala Gly Gly Ser Gly Arg Ala Gly 450 455 460 Pro Ala Pro Lys Gly Ser Ser Leu Gln Val Thr Phe Pro Ser Glu Thr 465 470 475 480 Leu Asn Leu Ser Glu Lys Cys Ile

Claims (11)

1. Claims: 1. Use of a gene selected from the group consisting of EMR3 and

   KMO as a biomarker for inflammatory diseases.
2. 2. Use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for macrophage activation.
3. 3. Use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for recruited macrophage activation.
4. 4. Use of a gene selected from the group consisting of EMR3 and KMO as a biomarker for an activated-macrophage-related disorder.
5. 5. The use according to claim 3, wherein said activated-macrophagerelated disorder is an inflammatory disorder.
6. 6. The use according to claim 4, wherein said inflammatory disorder is a respiratory inflammatory disorder.
7. 7. The use according to claim 5, wherein said respiratory inflammatory disorder is chronic obstructive pulmonary disease.
8. 8. A method of determining whether an individual suffers from CORD, wherein said method comprises the steps of: a) quantifying the macrophagic level of EMR3 or KMO expression and/or activity in a biological sample recovered from said individual; b) determining whether said macrophagic level of EMR3 or KMO expression and/or activity is elevated in said individual compared to the macrophagic level of EMR3 or KMO expression and/or activity in a control sample; and wherein said elevated level of macrophagic EMR3 or KMO expression and/or activity is taken as a sign of the presence of COPD.
9. 9. A method of screening for therapeutic agents useful in the treatment of inflammatory diseases comprising the steps of: 43.

   i) contacting a test compound with a polypeptide selected from the group consisting of EDG6 or PTGER4 polypeptides, ii) detect binding of said test compound to said polypeptide.
10. 10. A method of screening for therapeutic agents useful in the treatment of inflammation.

    diseases comprising the steps of: i) determining the activity of a polypeptide selected from the group consisting of EDG6 or PTGER4 polypeptides at a certain concentration of a test compound or in the absence of said test compound, ii) determining the activity of said polypeptide at a different concentration of said test compound.
11. 11. A method of diagnosing inflammatory diseases comprising the steps of: i) determining the amount of a polynucleotide selected from the group consisting of EDG6 or PTGER4 polypeptides in a sample taken from said mammal, ii) determining the amount of said polynucleotide in healthy and/or diseased mammals.